Regulation of the catalytic behaviour of L‐form starch phosphorylase from sweet potato roots by proteolysis

Abstract

Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the alpha-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the Km decreased during the proteolytic process when starch was used as the limiting substrate, but the Km for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (Vmax) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis.

Immobilization of starch phosphorylase from cabbage leaves: production of glucose-1-phosphate

“…They also showed that it played a role in the capacity of the leaf lumina to endure a transient water deficit. Chen et al (2002) showed regulation of the catalytic behavior of starch phosphorylase from sweet potato roots by proteolysis. They showed the presence of 78 amino acids in the center of the enzyme protein that served as a signal for rapid degradation.…”

Rice Endosperm Starch Phosphorylase (Pho1) Assembles with Disproportionating Enzyme (Dpe1) to Form a Protein Complex That Enhances Synthesis of Malto-oligosaccharides

“…In addition, like the L80 of OsPho1 the L78 of sweet potato, Pho1 has a number of charged amino acids that have been suggested to be potential targets for protein kinase activity (11). It was proposed that L78 serves as a switch controlling the catalytic direction of the enzyme reaction as proteolytic removal of the L78 region induced by the phosphorylation event resulted in alteration of the enzyme's affinities toward substrates (8).…”

“…The higher plant Pho1 is structurally homologous to Pho2 and microbial and animal glycogen phosphorylases with one significant exception. The enzyme from higher plants contains an additional 78 -80-amino acid peptide (L78/L80) positioned near the middle of the primary sequence, which is not found in Pho2, maltodextrin phosphorylase, or glycogen phosphorylases (4,8). This region, which has a number of charged amino acids, forms an undefined loop structure (supplemental Fig.…”

Immobilization of starch phosphorylase from seeds of Indian millet (Pennisetum typhoides) variety KB 560

“…They also showed that it plays a role in the capacity of the leaf lamina to endure a transient water deficit. Chen et al (2002) showed regulation of the catalytic behavior of starch phosphorylase from sweet potato roots by proteolysis. They showed the presence of 78 amino acids in the center of the enzyme protein that serves as a signal for rapid degradation.…”